PC-AssayDescription ::= {
aid {
id 651886,
version 1
},
aid-source db {
name "Southern Research Specialized Biocontainment Screening Center",
source-id str "VEE_VTR_05",
date std {
year 2013,
month 12,
day 1
}
},
name "Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV
(TC-83 strain) (5)",
description {
"Southern Research's Specialized Biocontainment Screening Center (SRSBSC)",
"Southern Research Institute (Birmingham, Alabama)",
"NIH Molecular Libraries Probe Production Centers Network (MLPCN)",
"Assay Provider: Dong Hoon Chung, University of Louisville",
"Award: 1 R03 MH087448-01A1 ",
"",
"Introduction:",
"Venezuelan equine encephalitis virus is one of the Alphavirus species in
the family of Togaviridae characterized by a non-segmented, positive sense
strand RNA virus (Griffin, D. E. 2001. Alphaviruses, p. 917-962. In D. Knipe
and P. M. Howley (ed.), Fields virology, vol. 4. Lippincott, Williams, and
Wilkins, Philadelphia, PA.). VEEV, endemic to North, Central and South
America, is an example of an arbovirus passing from a misquito vector through
rodent hosts and with epizootic subtypes causing disease in equines and
humans. While there are various subtypes, human infection tends to be acute
resulting in fever, headache, lymphopenia, myalgia and malaise but may
increase to severe encephalitis (Bale, J.F., Jr. 1993. Viral encephalitis.
Med Clin. North Am. 77:25-42.). Fatalities occur in ~1% of the population
(Steele, K., et al. 2007. Alphavirus encephalitides, p. 241-270. In Z. F.
Dembek (ed.), Medical aspects of biological warfare. Borden Institute (U.S.
Army Walter Reed), Washington, D.C.). Although recorded natural epidemics
are rare, the Centers for Disease Control and Prevention and the National
Institute of Allergy and Infectious Diseases have classified it as a category
B priority biodefense agent due to the ease at which it may be cultured,
manipulated and aerosolized. ",
"",
"In the United States, there are no licensed human vaccines for VEEV
although there is an Investigational New Drug (IND) vaccine given to at risk
populations. TC-83 is a live attenuated vaccine that provides long-lasting
immunity and protection. However ~25% of individuals vaccinated suffer from
adverse events and ~20% of those vaccinated do not produce an immune
response. C-84, derived from TC-83, is formalin-inactivated VEEV vaccine
that does not produce adverse reactions but conversely does not provide
protection against all VEE challenges and requires frequent boosting.
Currently there is no treatment for infection with VEE beyond supportive
care, so there is an intense need for potential therapeutics."
},
protocol {
"Biosafety and Biosecurity: All experiments with TC-83 strain was done in
the Regional Biocontainment Laboratory in University of Louisville. All
procedures were done in compliance with Select Agent Rules.",
" ",
"Cell Culture: Vero 76 cells (ATCC; CRL1586) were cultured in Dulbecco's
Modified Eagle's Medium (DMEM) with 4500 mg/L glucose, 2 mM L-glutamine, and
10% FBS (culture media). The cells are maintained at 37C, 5.0% CO2 to 100%
confluence being passaged every three to seven days. For cell plating, cells
were detached from flask bottom by using 0.05% Trypsin-EDTA solution and then
re-suspended in a growth media.",
"",
"VEEV culture: VEEV strain TC-83 was used for screening. The VEEV TC83
stock was prepared in BHK cells using an initial stock obtained from
USAMRIID. The stock was prepared in Vero 76 cells using an initial stock
obtained from World Reference Center for Emerging Viruses and Arboviruses
(Dr. Robert Tesh). Briefly, cells were grown in two T-175 flasks to 50%
confluence in a culture media. The cells were infected with 1mL of diluted
virus stock (1:10 dilution of the original stock) per T175 for 1.5 hours and
then washed, and replenished with 25mL media. The cells were incubated for 2
days in an incubator at 37C, 5% CO2 and high humidity. The supernatant was
harvested and the cell debris pelleted by centrifuging at 1,000 rpm for 5
minutes at 18C. The supernatant was aliquoted (1 ml per tube) and stored at
-80C. These virus stocks were titrated in Vero 76 cells using an agarose
overlay plaque method and the titers were 1.2XE09 pfu/ml.",
"",
"Cell Plating: Vero 76cells were seeded at 70% confluence in a 12-well
plate in a volume of 1mL and incubated for overnight at 37C with 5% CO2 and
high humidity. ",
"",
"Virus Addition: The cells were infected with virus by adsorption for an
hour. Cell culture media in the 12-well plates were removed completely and
the cells were infected with either mock virus (media only) or VEEV. VEEV
stock was diluted in the culture media to 9XE03 pfu/ml and 200uL was added to
the test wells and the virus control wells (final MOI of 0.1). The plates
were incubated in an actively humidified incubator with 5.0% CO2 at 37C for
one hour. During the incubation, plates were gently rocked every 20 min. to
ensure the coverage of the cells with virus. After the adsorption, the cells
were rinsed 1mL of PBS per well and then replenished with the media
containing testing articles. All additions were performed in a class II
Biosafety Cabinet. The plates were incubated in an actively humidified
incubator with 5.0% CO2 at 37C for 48 h. ",
"",
"Control and Drug Preparation: Carrier Control consisted of DMSO diluted
in assay media to 0.25% and 1000uL was dispensed to both cell and virus
control wells of 12- well tissue culture treated plates. Test compounds were
diluted in media to be at target concentration with a DMSO concentration of
0.25%.",
"",
"Titration of Progeny viruses (Mini plaque assay)",
"Titer of progeny viruses produced from the cell was measured by a mini
plaque assay in 96-well plate format. Fresh Vero 76 cells were seeded and
grown in 96-well plates overnight. The cell culture supernatants from the
96-well plates were emptied and the cells were infected with 25uL of 10-fold
serial dilutions of progeny virus containing medium from respective samples
(drug treated or untreated). The plates were incubated for one hour in an
incubator at 37C, 5% CO2. The cells were rinsed with 100uL per well of PBS
once and then replenished with DMEM with 0.75% methyl-cellulose and 10% FBS.
The cell plates were incubated at 37C, 5% CO2, and high humidity for an
additional three days. Crystal violet solution with 4% paraformaldehyde was
used to developed plaques in the wells. The assay plates were equilibrated to
room temperature for 10 minutes and then an equal volume of the crystal
violet solution was added to each well. The plates were incubated for 60 min
at room temperature and stained one more time. After a wash the plates with
water, the number of plaques in each wells were determined by a visual
counting. Virus titers were calculated by: No. of plaques X 10E (dilution
fold at the counting) * 1000 / 25 (pfu/mL). Compound treatment was done in a
duplicate independently and mean from duplicates of titration was used. Log
reduction of titer was calculated by: Log10(titer of Pos control) - Log10
(titer of sample)."
},
comment {
"Possible artifacts in this assay include, but are not limited to,
compounds that precipitate.",
"",
"Scoring: Of the compounds selected for dose response testing, those
showing more than 10 fold reduction in the progeny titer (